Many of the current recombinant therapeutic proteins are complex biopharmaceuticals that require manufacturing processes based on eukaryotic cell culture technology. While prokaryotic systems are also suited to produce certain recombinant therapeutic proteins at large scale, many of the more complex biopharmaceuticals are produced in eukaryotic cells, since these cells are able to support correct folding, processing and post-translational modification.
Any increase in volumetric productivity in eukaryotic cell culture has a direct impact on costs for these complex biopharmaceuticals and is, thus, desirable both from a commercial and healthcare socioeconomic view. The yield of any biopharmaceutical production process depends on the cumulative amount of protein product which the producing cells secrete per unit time when grown under regulated process conditions.
Several avenues have been pursued to boost production yields from mammalian cell culture with great success resulting in 10-20 fold yield increase over the past decade. Mammalian cell based production systems start to match with lower prokaryotic systems in volumetric yields while they still lag behind in fermenter occupancy times. Media and fed-batch process design allowed maintaining highest viable cell densities and have contributed significantly to this success.
The producer cell itself offers potential for improvement. All steps involved in the expression and secretion of heterologous proteins such as transcription, RNA transport, translation, post-translational modification and protein transport are tightly regulated and have a strong impact on the specific productivity. This complex pattern of features also including growth properties, low sensitivity to apoptosis and specific nutrient requirements provides the basis for the selection of existing cell lines for large scale production of biopharmaceuticals. While the cell lines were selected for serum and growth factor independence as well as suspension growth using genetic and epigenetic flexibility, molecular optimization focused on transcription first.
In contrast to artificial transcription factors or polymerases that did not provide a large benefit, protection from gene inactivation is considered essential. Promoter regions from housekeeping genes equipped for continuous expression or individual elements isolated from those promoters such as ubiquitous chromatin opening elements (UCOEs) and scaffold/matrix-associated regions (S/MARs) alter the chromatin structure of promoter regions, allowing free access by the polymerase II complex thereby reducing the number of clones to be screened and causing expression stability.
Nutrient depletion, waste byproduct accumulation, extended exposure to impeller shear forces and other factors that occur during fermentation constitute apoptotic stimuli which can decrease cell viability. Therefore, anti-apoptotic genes of the bcl-2 family (Kaufmann and Fussenegger, 2003) were intensively investigated. Since the integral of viable cell density (IVCD) is one of the factors that impact the overall volumetric productivity in mammalian cell culture, it is desirable to keep cell viability at a high level over the duration of upstream processing. Production processes with cell lines that overexpressed anti-apoptotic genes showed prolonged cell culture viability and occasionally increased product yields (Chiang and Sisk, 2005).
Another bottleneck for high-yield production of heterologous proteins in eukaryotic cells is the secretory transport machinery of the producer cell line. The expression and secretion of heterologous proteins in non-polarized eukaryotic cells is a complex regulated multi-step process with multiple actuating variables. In a first step, the gene encoding the secreted protein is transcribed into mRNA which is then exported into the cytosol. mRNAs encoding proteins destined for secretion into the extracellular space or to the plasma membrane are recognized by the signal recognition particle and transported to ribosomes located on the outer membrane of the rough endoplasmic reticulum. There, the nascent proteins are co-translationally imported into the luminal space of the endoplasmic reticulum where certain sorting mechanisms apply. From there, they are packed in lipid vesicles and transported to the proximal cisternae of the Golgi apparatus and finally they are packaged into vesicles emerging from the distal trans-Golgi network (TGN) which are then transported to the plasma membrane where they are released into the culture medium. The entire secretion process is not straightforward but highly regulated on various levels. Known checkpoints and feedback loops of the secretion pathway include the Calnexin/Calreticulin System, the Unfolded protein response and ER overload response.
As a logical consequence of this complexity, the elimination of one bottleneck at early process stages is likely to lead to the creation of other bottlenecks either further downstream in the process chain or even in other crucial parallel sidelines of the secretion process. There is evidence in the literature that widening of the transcription and translation bottleneck is only effective up to a certain threshold. The specific productivity of a producer cell line has been reported to correlate linearly with the level of product gene transcription until a certain threshold mRNA-level is reached (Barnes et al., 2007). Further enhancement of transcriptional activity to mRNA levels beyond this threshold does not cause further titer increment. Rather, productivity even goes down as translation from the excess mRNA causes an overload of the protein synthesis, folding or transport machinery which in turn results in an intracellular accumulation of the protein product and concurrent triggering of the ER-Overload- and Unfolded Protein Response. As one of its consequences, the alpha subunit of the polypeptide chain initiation factor eIF2 is phosphorylated. The phosphorylated eIF2α inhibits the guanine nucleotide exchange factor eIF2β and prevents its recycling during protein synthesis. As a result, the overall rate of protein synthesis is diminished.
Specific differentiated cells such as plasma cells show very high protein secretion rates. This capacity is acquired during the terminal differentiation of B cells and has been associated with the expression of a splice form of the transcription factor XBP (Iwakoshi et al., 2003). XBP-1 regulates this differentiation process by binding to ER stress responsive elements (ERSE) within the promoters of a wide spectrum of secretory pathway genes. The XBP-1 mediated differentiation process results in a physical expansion of the ER, increased mitochondrial mass and function, larger cell size and enhanced total protein synthesis (Shaffer et al., 2004). This complex functional change is associated with cell cycle arrest.
Almost all cell types involved in high-level protein secretion (pancreatic beta cells, leydig cells, glandular cells, etc.) are terminally differentiated, are not able to proliferate and have a limited life-span before ultimately undergoing programmed cell death (Chen-Kiang, 2003). Unsurprisingly, attempts to increase protein secretion by overexpressing XBP-1 in non-plasma cells, especially production cell lines such as CHO-K1 cells, were only successful in increasing protein secretion to a limited extent (Tigges and Fussenegger, 2006). A high proliferation rate during early process phases and high cell viability remain critical for modern fed-batch production processes.
This general paradigm holds true for other factors upstream of XBP-1 capable of enhancing the secretory capacity such as ATF6 and IRE1α. Higher cell specific productivity due to enhanced secretion needs to compensate for lower viable cell mass and shorter process duration before higher yields are feasible. Moreover, expression of growth inhibitory genes is difficult to maintain and process robustness may suffer.
Molecular chaperones such as binding protein BiP/GRP78 and protein disulfide isomerase (PDI) are downstream effectors of XPB. BiP associates with the antibody heavy chain before its displacement by the light chain and plays a major role in its folding. Both BiP/GRP78 and protein disulfide isomerase (PDI) have been considered for secretion enhancement but were not yet successful in improving upstream yield. In contrast to what could be expected, BiP overexpression in mammalian cells has been shown to reduce rather than to increase the secretion of proteins with which BiP associates with (Domer and Kaufman, 1994). Likewise, PDI overexpression in CHO cells reduced the expression of a TNFR:FC fusion protein (Davis et al., 2000), whereas the specific production rate of an antibody could be increased by 40% (Borth et al., 2005).
Thus, there are different bottlenecks within the protein secretion machinery which are not yet overcome.
Further, the fact that an increase of the protein folding capacity of a cell creates a protein production bottle neck further downstream, is supported by a report describing ER to cis-Golgi transport problems for IFN-gamma production in a CHO cell line (Hooker et al., 1999). Other additional bottlenecks are expected further downstream in the cellular secretion machinery.
All of the above-described examples show that multiple bottlenecks, for example, within the protein production machinery and/or protein secretion machinery, exist which have to be overcome in order to optimize the preparation of secreted proteins in cells.
Thus, there is a need for protein producing cells comprising a regulator protein having an effect on multiple levels of cellular function. Said effect on multiple levels of cellular function should result in an enhancement of protein production and/or protein secretion, particularly in large-scale industrial protein production processes, e.g. industrial fed batch processes. There is also a need for a method for producing proteins (e.g. therapeutic proteins) using said cells.
Systems biology approaches, in particular comparative transcriptome and proteome analysis of high and low expressing cells, may identify molecular targets that help to overcome productivity bottlenecks. However, the combined expression of multiple regulators in a well adjusted and stable fashion is rather complex and may not be practical in all cases. On the other hand, the expression of individual high level regulators is an approach that allows to affect multiple pathways simultaneously. Nevertheless, the pleiotropic effects of such active high level regulator proteins and their mutants makes it difficult to predict how their expression within the cellular context will affect upstream product yield in industrial fed batch processes.
Many approaches for yield-enhancement by cell line engineering have focused on single enzymes and regulator proteins in the past. Only few attempts have been made to investigate the potential of regulator proteins that are known for their pleiotropic effects on multiple levels of cellular function. To some extent, the lack of research in this field may have resulted from the contradictive findings as reported in the literature and as described above.
One protein family that contains known high level regulators with pleiotropic effector functions is the GTPase superfamily.
The superfamily of small GTP binding proteins contains more than 100 members that can be divided into five groups, namely into the Ras, Rho, Rab, Sar/Arf and Ran group, based on structural and functional differences. Whereas regulation of gene expression has initially been associated only with the Ras proteins and vesicular transport with the Sar/Arf proteins, the Rho proteins were thought to control the organization of the cytoskeleton (Takai et al., 2001). It has later been shown that besides actin organization the three major members of the Rho family, namely Rho, cdc42 and Rac, are involved in processes of cell cycle progression, transcription, cell-cell adhesion, cell motility, vesicle shuttling, secretion, endocytosis, phagocytosis, mitogenesis, and/or apoptosis. This pleiotropic activity makes the Rho GTPases to interesting candidates as general regulators of cellular processes. However, only part of the natural responses will have the technically desired direction. For example, cell cycle promotion may induce proapoptotic signals, while differentiation may lead to ER expansion and enhanced secretory capacity.
Rho GTPases exist in an inactive GDP-bound and an active GTP-bound form. In their active conformation they can interact with a large number of effectors regulating multiple signal transduction pathways. Activation of Rho GTPases is mediated by guanine nucleotide exchange factors (GEFs) which catalyze the replacement of GDP by GTP. Only a fraction of the more than 80 GEFs known to date have been characterized in detail, but it is clear that many GEFs can activate more than one Rho GTPase (Schmidt and Hall, 2002). The activity of the Rho proteins, including cdc42, depends on their GTPase cycle. Said proteins are active in their GTP-bound form and inactive in their GDP-bound form. Three types of regulatory proteins control said GTPase cycle: (i) the Guanine nucleotide exchange factors (GEFs), (ii) the GTPase activating proteins (GAPs), and (iii) the Guanine nucleotide dissociation inhibitors (GDIs). The complexity of the functional network of Rho GTPases may provide an explanation for the seemingly contradictive effects of Rho GTPase overexpression that has been reported in the literature.
The GEF proteins that control Rho GTPases are tightly regulated themselves by inhibitory domains or inhibitor proteins and are activated upon dephosphorylation. Members of the dbl family of oncogenes that are overexpressed in a variety of cancer types represent guanine nucleotide exchange factors (GEFs) for cdc42 that help to maintain cdc42 in its activated state. This may led to the assumption that permanently active (GTP-bound) cdc42 could activate cell growth, and other functions attributed to cdc42. While heterologous overexpression of a tightly regulated protein is expected to have little functional impact, overexpression of a permanently activated regulator protein maintained in its activated state would be desirable. For cdc42, the permanently active mutants cdc42Q61L and cdc42V12 have been isolated. These mutants were shown to have positive effects on total secretion rates of p75, while LDLR secretion was inhibited in MDCK cells (Müsch et al., EMBO J. 2001 May 1; 20(9): 2171-2179). Efficient movement of correctly folded proteins from the ER to the Golgi is considered as an essential step in the production of secreted proteins. Contrary to expectations, it was found that expression of a permanently active GTPase-defective Cdc42 mutant led to growth inhibition or apoptosis. Moreover, the same constitutively active mutant cdc42Q61L was shown to block the ER to Golgi transport of VSVG (Wu 2000 Nature).
To resolve the scientifically contradictory results and to obtain cdc42s which are more active than the natural cdc42s, fast cycling cdc42 mutants such as cdc42F28L (R. Lin et al., A novel Cdc42Hs mutant induces cellular transformation. Curr. Biol. 7 (1997), pp. 794-797) or cdc42D118N (S. Tu et al., Antiapoptotic Cdc42 mutants are potent activators of cellular transformation. Biochemistry 41 (2002), pp. 12350-12358) have been generated. These mutants can undergo spontaneous GTP-GDP exchange while maintaining full GTPase activity. These mutants are no longer dependent on induction by dbl. Said mutants are self-activating mutants. The fast cycling cdc42 mutants such as cdc42F28L do not block trafficking as the permanently active cdc42 mutants (Wu 2000 Nature), but cause moderate acceleration. The cdc42F28L mutant, for example, survived in NIH 3T3 fibroblasts and allowed anchorage independent growth and increased growth rates in media with reduced serum (Tu et al., 2002). However, increased growth rates may contradict a high productivity as it is generally accepted that fast replication does not allow to build up the membrane systems required for efficient secretion and folding.
Cdc42 appears as one of the very broad and heterogeneous regulators of cellular activity. Nevertheless, as discussed above, the natural, permanently active and inactive forms of cdc42 have demonstrated features incompatible with a production cell. So far, cdc42 mediated active actin reorganization, fast cycling of vesicles and/or induction of anchorage independence has/have not been linked with increased productivity of non-adherent cells.
In a screen under industrial protein production process relevant conditions, the inventors of the present invention surprisingly found that fast cycling cdc42 mutants exert a net beneficial effect on protein yields obtained in non-adherent cell cultures. Said fast cycling cdc42 mutants directly boost productivity of non-adherent cell cultures, e.g. in a bioreactor setting. In particular, the fast cycling cdc42 mutants accelerate trafficking of proteins from the ER to the Golgi. As mentioned above, efficient movement of correctly folded proteins from the ER to the Golgi is considered as an essential step in the production of secreted proteins.
The inventors of the present invention further found that the beneficial effect of the fast cycling cdc42 mutants on protein yields is independent of the order in which the heterologous genes are introduced into the non-adherent cells. For example, said effect is achieved when (i) a high producer cell clone expressing the protein of interest is subsequently transfected with a nucleic acid sequence encoding a fast cycling cdc42 mutant, (ii) a naïve non-adherent cell is modified with a fast cycling cdc42 mutant to create a superior starter cell that is subsequently transfected with a nucleic acid sequence encoding the protein of interest or (iii) a naïve non-adherent cell is transfected simultaneously with a nucleic acid sequence encoding a fast cycling cdc42 mutant and with a nucleic acid sequence encoding the protein of interest.